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Downregulation of the taurine transporter TauT during hypo-osmotic stress in NIH3T3 mouse fibroblasts.

Hansen DB, Friis MB, Hoffmann EK, Lambert IH - J. Membr. Biol. (2012)

Bottom Line: Taurine influx is reduced following reduction in osmolarity, keeping the extracellular Na(+) concentration constant.TonEBP activity is unaltered, whereas TauT transcription as well as TauT activity are significantly reduced under hypo-osmotic conditions.Acute exposure to ROS reduces taurine uptake as a result of modulated TauT transport kinetics.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Section of Cellular and Developmental Biology, University of Copenhagen, The August Krogh Building, Universitetsparken 13, 2100 Copenhagen Ø, Denmark.

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Effect of long-term hyper- and hypo-osmotic conditions on TonEBP activity and TauT transcription. TonEBP activity and TauT transcription were estimated in NIH3T3 cells exposed to iso-osmotic, hyperosmotic or hypo-osmotic medium (DMEM) for 16 and 4 h, respectively. a For estimation of TonEBP activity, cells were transfected with luciferase-plasmid and incubated with the indicated medium and luciferase activity was estimated as indicated in Materials and Methods. b TauT mRNA transcription was estimated by qPCR. cDNA was generated from mock-transfected NIH3T3 cells, and qPCR was performed using primers specific for TauT mRNA as indicated in Materials and Methods. Values are given relative to their respective iso-osmotic control ± SEM. Data in (a) represent seven sets of paired experiments. Data in (b) represent four and five sets of paired experiments for hyperosmotic and hypo-osmotic, respectively. Statistical evaluation for (a) and (b) by Student’s t-test (paired, one-sided) comparing hyperosmotic or hypo-osmotic to iso-osmotic control, respectively. *P < 0.05, **P < 0.01 compared to iso-osmotic control
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Fig2: Effect of long-term hyper- and hypo-osmotic conditions on TonEBP activity and TauT transcription. TonEBP activity and TauT transcription were estimated in NIH3T3 cells exposed to iso-osmotic, hyperosmotic or hypo-osmotic medium (DMEM) for 16 and 4 h, respectively. a For estimation of TonEBP activity, cells were transfected with luciferase-plasmid and incubated with the indicated medium and luciferase activity was estimated as indicated in Materials and Methods. b TauT mRNA transcription was estimated by qPCR. cDNA was generated from mock-transfected NIH3T3 cells, and qPCR was performed using primers specific for TauT mRNA as indicated in Materials and Methods. Values are given relative to their respective iso-osmotic control ± SEM. Data in (a) represent seven sets of paired experiments. Data in (b) represent four and five sets of paired experiments for hyperosmotic and hypo-osmotic, respectively. Statistical evaluation for (a) and (b) by Student’s t-test (paired, one-sided) comparing hyperosmotic or hypo-osmotic to iso-osmotic control, respectively. *P < 0.05, **P < 0.01 compared to iso-osmotic control

Mentions: TauT transcription in mammalian cells is upregulated by TonEBP under hyperosmotic conditions (Miyakawa et al. 1998, 1999b), and we have previously shown that 4 h hyperosmotic exposure increases TauT activity in, e.g., NIH3T3 cells (Voss et al. 2004). As the effects of hypo-osmotic exposure on taurine uptake appeared to be acute and independent of TauT transcription, we tested whether TonEBP activity and TauT transcription were actually unaffected by prolonged exposure to hypo-osmotic conditions. From Fig. 2 it is seen, in accordance with previous data, that exposure of NIH3T3 cells to hyperosmotic conditions results in a significant 16-fold increase in TonEBP activity within 16 h (Fig. 2a) and an almost twofold increase in TauT mRNA within 4 h (Fig. 2b). However, TonEBP activity is unaffected by 16 h hypo-osmotic exposure (Fig. 2a), which is in agreement with previously demonstration of a reduction in TonEBP mRNA and retention of TonEBP in the cytoplasm under hypo-osmotic conditions (Woo et al. 2000). However, despite the unaffected TonEBP activity, TauT mRNA is reduced after 4 h hypo-osmotic exposure (Fig. 2b). A selection of cells expressing low TauT as the cause of the observed data is most unlikely as kinetic analysis (Voss et al. 2004) revealed that there is only one population of NIH3T3 cells (one Km value for taurine) and that the time frame of our experiments is very short. Hence, TauT mRNA levels correlate with TonEBP activity under long-term hyperosmotic conditions but not under long-term hypo-osmotic conditions.Fig. 2


Downregulation of the taurine transporter TauT during hypo-osmotic stress in NIH3T3 mouse fibroblasts.

Hansen DB, Friis MB, Hoffmann EK, Lambert IH - J. Membr. Biol. (2012)

Effect of long-term hyper- and hypo-osmotic conditions on TonEBP activity and TauT transcription. TonEBP activity and TauT transcription were estimated in NIH3T3 cells exposed to iso-osmotic, hyperosmotic or hypo-osmotic medium (DMEM) for 16 and 4 h, respectively. a For estimation of TonEBP activity, cells were transfected with luciferase-plasmid and incubated with the indicated medium and luciferase activity was estimated as indicated in Materials and Methods. b TauT mRNA transcription was estimated by qPCR. cDNA was generated from mock-transfected NIH3T3 cells, and qPCR was performed using primers specific for TauT mRNA as indicated in Materials and Methods. Values are given relative to their respective iso-osmotic control ± SEM. Data in (a) represent seven sets of paired experiments. Data in (b) represent four and five sets of paired experiments for hyperosmotic and hypo-osmotic, respectively. Statistical evaluation for (a) and (b) by Student’s t-test (paired, one-sided) comparing hyperosmotic or hypo-osmotic to iso-osmotic control, respectively. *P < 0.05, **P < 0.01 compared to iso-osmotic control
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Fig2: Effect of long-term hyper- and hypo-osmotic conditions on TonEBP activity and TauT transcription. TonEBP activity and TauT transcription were estimated in NIH3T3 cells exposed to iso-osmotic, hyperosmotic or hypo-osmotic medium (DMEM) for 16 and 4 h, respectively. a For estimation of TonEBP activity, cells were transfected with luciferase-plasmid and incubated with the indicated medium and luciferase activity was estimated as indicated in Materials and Methods. b TauT mRNA transcription was estimated by qPCR. cDNA was generated from mock-transfected NIH3T3 cells, and qPCR was performed using primers specific for TauT mRNA as indicated in Materials and Methods. Values are given relative to their respective iso-osmotic control ± SEM. Data in (a) represent seven sets of paired experiments. Data in (b) represent four and five sets of paired experiments for hyperosmotic and hypo-osmotic, respectively. Statistical evaluation for (a) and (b) by Student’s t-test (paired, one-sided) comparing hyperosmotic or hypo-osmotic to iso-osmotic control, respectively. *P < 0.05, **P < 0.01 compared to iso-osmotic control
Mentions: TauT transcription in mammalian cells is upregulated by TonEBP under hyperosmotic conditions (Miyakawa et al. 1998, 1999b), and we have previously shown that 4 h hyperosmotic exposure increases TauT activity in, e.g., NIH3T3 cells (Voss et al. 2004). As the effects of hypo-osmotic exposure on taurine uptake appeared to be acute and independent of TauT transcription, we tested whether TonEBP activity and TauT transcription were actually unaffected by prolonged exposure to hypo-osmotic conditions. From Fig. 2 it is seen, in accordance with previous data, that exposure of NIH3T3 cells to hyperosmotic conditions results in a significant 16-fold increase in TonEBP activity within 16 h (Fig. 2a) and an almost twofold increase in TauT mRNA within 4 h (Fig. 2b). However, TonEBP activity is unaffected by 16 h hypo-osmotic exposure (Fig. 2a), which is in agreement with previously demonstration of a reduction in TonEBP mRNA and retention of TonEBP in the cytoplasm under hypo-osmotic conditions (Woo et al. 2000). However, despite the unaffected TonEBP activity, TauT mRNA is reduced after 4 h hypo-osmotic exposure (Fig. 2b). A selection of cells expressing low TauT as the cause of the observed data is most unlikely as kinetic analysis (Voss et al. 2004) revealed that there is only one population of NIH3T3 cells (one Km value for taurine) and that the time frame of our experiments is very short. Hence, TauT mRNA levels correlate with TonEBP activity under long-term hyperosmotic conditions but not under long-term hypo-osmotic conditions.Fig. 2

Bottom Line: Taurine influx is reduced following reduction in osmolarity, keeping the extracellular Na(+) concentration constant.TonEBP activity is unaltered, whereas TauT transcription as well as TauT activity are significantly reduced under hypo-osmotic conditions.Acute exposure to ROS reduces taurine uptake as a result of modulated TauT transport kinetics.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Section of Cellular and Developmental Biology, University of Copenhagen, The August Krogh Building, Universitetsparken 13, 2100 Copenhagen Ø, Denmark.

Show MeSH
Related in: MedlinePlus